Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 A.

A multitude of heptahelical receptors use heterotrimeric G proteins to transduce signals to specific effector target molecules. The G protein transducin, Gt, couples photon-activated rhodopsin with the effector cyclic GMP phosophodiesterase (PDE) in the vertebrate phototransduction cascade. The interactions of the Gt alpha-subunit (alpha(t)) with the inhibitory PDE gamma-subunit (PDEgamma) are central to effector activation, and also enhance visual recovery in cooperation with the GTPase-activating protein regulator of G-protein signalling (RGS)-9 (refs 1-3). Here we describe the crystal structure at 2.0 A of rod transducin alpha x GDP x AlF4- in complex with the effector molecule PDEgamma and the GTPase-activating protein RGS9. In addition, we present the independently solved crystal structures of the RGS9 RGS domain both alone and in complex with alpha(t/i1) x GDP x AlF4-. These structures reveal insights into effector activation, synergistic GTPase acceleration, RGS9 specificity and RGS activity. Effector binding to a nucleotide-dependent site on alpha(t) sequesters PDEgamma residues implicated in PDE inhibition, and potentiates recruitment of RGS9 for hydrolytic transition state stabilization and concomitant signal termination.

Prediction and confirmation of a site critical for effector regulation of RGS domain activity.

A critical challenge of structural genomics is to extract functional information from protein structures. We present an example of how this may be accomplished using the Evolutionary Trace (ET) method in the context of the regulators of G protein signaling (RGS) family. We have previously applied ET to the RGS family and identified a novel, evolutionarily privileged site on the RGS domain as important for regulating RGS activity. Here we confirm through targeted mutagenesis of RGS7 that these ET-identified residues are critical for RGS domain regulation and are likely to function as global determinants of RGS function. We also discuss how the recent structure of the complex of RGS9, Gt/i1alpha-GDP-AlF4- and the effector subunit PDEgamma confirms their contact with the effector-G protein interface, forming a structural pathway that communicates from the effector-contacting surface of the G protein and RGS catalytic core domain to the catalytic interface between Galpha and RGS. These results demonstrate the effectiveness of ET for identifying binding sites and efficiently focusing mutational studies on their key residues, thereby linking raw sequence and structure data to functional information.

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability.

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.

Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast.

Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo. Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro. A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone. Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchange chromatography. Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations. These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.